EP1774023B1 - Marker für hirnschädigung - Google Patents

Marker für hirnschädigung Download PDF

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EP1774023B1
EP1774023B1 EP05763857.9A EP05763857A EP1774023B1 EP 1774023 B1 EP1774023 B1 EP 1774023B1 EP 05763857 A EP05763857 A EP 05763857A EP 1774023 B1 EP1774023 B1 EP 1774023B1
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gene
encoding
protein
brain
antibody
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EP1774023A4 (de
EP1774023A2 (de
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Jack Ladenson
Yvonne Landt
Vijay Modur
Omar Laterza
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University of Washington
Washington University School of Medicine
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Washington University School of Medicine
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the invention relates to a method to diagnose brain damage such as stroke or Alzheimer's disease in humans and corresponding conditions in other animals. More specifically, it concerns methods to identify brain damage markers, and methods to diagnose brain damage using these markers and materials for their detection.
  • brain damage such as that caused by stroke or Alzheimer's disease.
  • Early diagnosis of, for example, ischemic stroke is believed critical in order to permit an appropriate intervention, such as administration of recombinant tissue plasminogen activator which has been shown to he highly effective, if administered early, in reducing mortality and morbidity resulting from stroke.
  • other forms of brain damage such as hemorrhagic stroke, damage to asphyxiated term infants, brain damage resulting from cardiac surgery, Alzheimer's or miscellaneous neurodegenerative disorders are desired to be assessed; although additional diagnostic procedures may be required to distinguish among these various possibilities in some cases.
  • CSF cerebrospinal fluid
  • serum or plasma or urine proteins or other substances that are generally characteristic of the brain.
  • the desirability of identifying such factors that could be used for diagnoses so as to identify appropriate treatment or simply for prognosis has also been recognized widely. See, for example, Warlow, C., Lancet (2003) 362:1211-1224 ; Qureshi, A., et al., New Eng J Med, (2001) 344:1450-1460 ; Marler, J.
  • S-100B is a Ca 2+ -binding protein that modulates complex neuronal-glial interactions and is found mostly in glia, melanocytes, Schwann cells, Langerhans cells and anterior pituitary cells, but not in neurons. Elevated serum levels of S-100B have been associated with stroke, post-cardiac arrest brain injury and traumatic head injury.
  • Aurell, A et al., Stroke (1991) 22:1254-1258 ; Hardemark, H. G., et al., J Neurosurg (1989) 71:727-731 ; Noppe, M, et al., Clin Chim Acta (1986) 155:143-150 ; Bottiger, B.
  • S-100B and NSE are significant markers of brain damage is set forth in Mussack, T., et al., Shock (2002) 18:395-400 and in a comment on this article by Vos, P. F. et al., ibid. 481-482 . It is noted that increased serum concentrations of S-100B, GFAP, and NSE have been associated with various acute central nervous system disorders.
  • S-100B is not brain specific ( Vaage, J., et al., J Thorac Cardiovasc Surg (2001) 122:853-855 ; Unden, J., Scand J Infect Dis (2004) 36:10-13 ) since it is also expressed in white and brown adipose tissue, skin, skeletal muscle, melanoma and glioblastoma cells ( Zimmer, D. B., et al., Brain Res Bull (1995) 37:417-429 ; Ilg, E.
  • NSE represents the gamma, gamma-dimer of the protein enolase (2-phospho-D-glycerate hydrolase), which is a soluble enzyme of the glycolytic pathway with a total molecular weight of approximately 80 kDa ( Schmechel, D., et al., Science (1978) 199:313-315 ). NSE is expressed in neuronal cytoplasm and dendrites and in cells of the amine precursor uptake and decarboxylation (APUD) cell system.
  • APUD amine precursor uptake and decarboxylation
  • Combinations of markers have been used in an attempt to obtain better sensitivity and specificity for stroke.
  • One group has utilized the combination of brain markers neuron-specific enolase, myelin basic protein, and S-100B ( Kupchak, P., et al., Clin Chem (2005) 51(6):A119 and A120 ; abstracts).
  • Another group evaluated >50 protein biomarkers and chose S-100B, B-type neurotrophic growth factor, Von Willebrand factor, matrix metalloproteinase-9 and monocyte chemotactic protein-1 ( Reynolds, M., Clin Chem (2003) 45(10):1733-1739 ).
  • biomarkers based on brain damage S100B
  • inflammation matrix metalloproteinase-9 and vascular cell adhesion molecule
  • thrombosis Von Willebrand factor
  • the markers are S-100B, brain natriuretic protein, D-dimer, and matrix metalloproteinase-9.
  • WO 2004/053052 A discloses a method of diagnosing the occurrence of brain damage, wherein fresh brain tissue is used, and the expression of specific markers is determined, and wherein a over-expression of these markers indicates the existence of brain damage.
  • Bernstein et al. ( Bernstein H.G., Journal of Neurocytology (1999) 28: 655 - 662 ) discloses the determination of the expression and different immunoreactivities of visinin-like-1 in the human brain, wherein antibodies are used.
  • the description provides methods to identify markers of brain damage and methods to predict the presence and progression of brain damage using these markers.
  • the markers are proteins or their encoding mRNA's that demonstrate enhanced expression of the relevant gene in the brain.
  • body fluids e.g., in cerebrospinal fluid, blood, or in urine they are associated with the tissue damage characteristic of brain damage.
  • a particularly important form of brain damage is stroke, which affects large numbers of individuals and, if correctly identified sufficiently early, permits effective treatment. It is estimated that many stroke victims die or exhibit vastly decreased quality of life due to the inability of currently used approaches correctly to diagnose and treat the condition in sufficient time.
  • Other indications where brain damage is significant include but are not limited to trauma, damage due to asphyxiation, damage associated with invasive surgery, and neurodegenerative diseases such as Alzheimer's.
  • the invention is directed to a method to identify visinin-like 1 (VLP-1) useful in brain damage diagnosis which method comprises assessing fresh or fresh-frozen brain tissue for gene expression and comparing the levels of gene expression in said fresh or fresh-frozen brain tissue with expression levels of the same genes in other fresh or fresh-frozen tissues, such as muscle or liver.
  • VLP-1 with expression levels higher in fresh brain than in alternative tissues is identified as generating mRNA or protein markers for brain damage.
  • markers are 1) genes that show expression levels of mRNA at least 10,000 times background in brain tissue; 2) genes that encode proteins of ⁇ 70 kD molecular weight; and 3) instances in which the protein encoded by the gene is detectable in the brain using Western blot or other standard techniques. Expression at high levels above background assures sufficient abundance in the brain for the expression markers to be useful as detection targets.
  • the method employ fresh or fresh-frozen brain tissue as could be derived from laboratory animals such as mice, rats or rabbits since the levels of mRNA and protein in brain (or other tissues) can be altered when the tissue is stored improperly, or in the case of direct testing on human brains, when the brain is in an unnatural state as would be necessarily the case in autopsied tissue.
  • this surrogate species There is no detriment to utilizing this surrogate species, as human analogs of the markers determined in fresh animal tissue can readily be identified.
  • antibodies, including monoclonal antibodies are prepared that are cross-reactive with the markers identified in laboratory animals and their human counterparts.
  • a method to diagnose the occurrence of, or a high probability of subsequent occurrence of, brain damage in a subject comprises analyzing biological fluids of said subject for the presence of LP-1 and one or more markers identified as described above.
  • markers include, but are not limited to, the products of genes located at gene I.D. No. 7447 (VSNL1) encoding visinin-like 1 (VLP-1), gene I.D. No. 6616 (SNAP25) encoding synaptosomal-associated protein, 25kDa, gene I.D. No. 2571 (GAD1) encoding glutamate decarboxylase 1 (brain, 67 kDa (GAD67), gene I.D. No.
  • MOBP myelin-associated oligodendrocyte basic protein
  • SYT1 synaptotagmin I
  • TUBB4 tubulin, beta 4
  • FEZ1 fasciculation and elongation protein zeta 1 (zygin I)
  • GLRB glycine receptor
  • VMP vesicular membrane protein p24
  • OLM1 olfactomedin 1, gene I.D.
  • ZIC1 Zic family member 1 (odd-paired homolog, Drosophila), gene I.D. No. 29993 (PACSIN1) encoding protein kinase C and casein kinase substrate in neurons 1, gene I.D. No. 5354 (PLP1) encoding Proteolipid protein 1 (Pelizaeus-Merzbacher disease, spastic paraplegia 2), gene I.D. No. 9118 (INA) encoding internexin neuronal intermediate filament protein, alpha, gene I.D. No. 140679 (SLC32A1) encoding solute carrier family 32 (GABA vesicular transporter), member 1, gene I.D. No.
  • SERPINI1 serine (or cysteine) proteinase inhibitor, clade I (neuroserpin), member 1, gene I.D. No. 4826 (NNAT) encoding Neuronatin, gene I.D. No. 2566 (GABRG2) encoding gamma-aminobutyric acid (GABA) A receptor, gamma 2, gene I.D. No. 6844 (VAMP2) encoding vesicle-associated membrane protein 2 (synaptobrevin 2), or gene I.D. No. 4900 (NRGN) encoding neurogranin (protein kinase C substrate, RC3).
  • GABA gamma-aminobutyric acid
  • VAMP2 vesicle-associated membrane protein 2
  • NRGN encoding neurogranin (protein kinase C substrate, RC3).
  • compositions useful in determining the presence or levels of the markers of the invention in biological fluids of subjects to be diagnosed include antibodies specifically immunoreactive with the proteins encoded by the relevant genes, oligonucleotide probes specifically hybridizing under stringent conditions to the mRNA transcribed from these genes, and oligonucleotide primers appropriate to amplify said mRNA.
  • kits for diagnosis which contain antibodies, oligonucleotide probes, and/or primers and other reagents for identifying association of these reagents with targets in the biological fluid samples.
  • Figures 1A and 1B show the comparative mRNA levels of 20 brain damage markers in brain and other tissues.
  • Figure 2 shows the results of Western blot of brain tissue homogenate demonstrating the presence of the protein expression products of genes VSNL-1, FEZ1, SERPINI1 and GAD1.
  • Figure 3 shows Western blots demonstrating the presence of the protein expression product of genes INA, SNAP25, and MOBP. PLP-1 did not show protein expression detectable by this method.
  • Figures 4A and 4B show standard curves for the VLP-1 sandwich assay.
  • Figures 5A-5E show the results of determination of VLP-1 protein in the plasma of blood samples taken from patients diagnosed as having suffered ischemic stroke.
  • Figure 6 shows the results of assays of rat serum in a rat stroke model assayed for VLP- 1.
  • Figures 7A-7C show standard curves for sandwich assays developed for neuroserpin, GAD67 and zygin, respectively.
  • Fresh brain tissue can be obtained from experimental laboratory animals, such as rabbits, rats, mice, guinea pigs, and the like. (As used herein, "fresh” includes fresh frozen.)
  • a preferred source is fresh brain tissue from mice.
  • the non-brain tissue used for comparison is preferably fresh as well and obtained from the same subject.
  • expression is detected by utilizing gene microarrays; other methods of assessing expression are also applicable, including quantitative RT-PCR, immunoassays for protein, and the like. Methods to assess gene expression are well known in the art and many such methods are commercially available.
  • genomic loci As set forth above, a number of genomic loci have been identified as highly expressed in brain, but at much lower levels, or not at all in other tissues and organs. Transit of the expression products of these loci from the brain itself into biological fluids is verified as associated with brain damage or the probability of, for example, stroke onset.
  • antibodies have been prepared that are specifically immunoreactive with some of the proteins identified. These antibodies, as well as other specific binding partners, are useful in methods for stroke diagnosis by detecting proteins in biological fluids.
  • a primary criterion for identifying a gene as generating an mRNA or protein marker for brain damage is specificity of expression in the brain.
  • genes were chosen whose mRNA expression levels in brain were at least 10 times higher than expression levels in a variety of other tissues including lung, intestine, skeletal muscle, pancreas, heart, liver, spleen and kidneys. Additional criteria are also helpful. It is useful to assure an expression level in the brain that will provide sufficient target for detection in the assay.
  • This criterion is referred to as the level of expression as compared to background.
  • the background is defined as signals obtained from mismatch oligonucleotides in a GeneChip ® array format.
  • the average difference values for the mRNA generated by the gene in question are the difference between the signal intensity of the appropriate RNA (generated when a perfect match occurs) and the signal created when mismatch oligonucleotides are employed. Details of this criterion are set forth in Example 1. Genes that give mRNA expression with levels more than 10,000-fold above said background are considered sufficiently abundant in the brain to provide a basis for detection.
  • assays are to be conducted at the protein level, it is desirable to assure the presence of adequate levels of proteins by assaying brain tissue homogenates using Western blot. Most of the genes identified as markers in the present application provide detectable levels of protein from brain homogenates on Western blot. Further, if assays at the protein level are employed, it is desirable to select genes that encode proteins of ⁇ 70 kDa to assure ability to pass through the blood-brain barrier.
  • the first two criteria (10-fold higher expression in the brain as compared to other tissues) and a 10,000-fold higher level of expression versus background are particularly significant when mRNA is used as a marker, and the appearance of protein on Western blot and selection of proteins of ⁇ 70 kDa are particularly important when protein is used as the marker.
  • Biological fluids that are useful as samples for the detection methods of the invention include blood and fractions thereof, such as plasma or serum, cerebrospinal fluid, urine, and lymphatic fluids, Serum or plasma or urine are more convenient.
  • the subjects may be human patients, or may be animals such as domesticated companion animals, farm animals, experimental laboratory animals, or any vertebrate system subject to brain damage.
  • the subjects may be humans, cows, horses, pigs, cats, dogs, rats, rabbits, mice, chickens and other fowl, or horses. Of greatest interest is detection of these markers in biological fluids of humans.
  • a wide variety of methods to detect gene expression in biological fluids is available.
  • a very brief exemplary list would include detection of mRNA levels using quantitative PCR with oligonucleotide primers as reagents, Northern blot or other formats using oligonucleotide probes that hybridize specifically to mRNA transcribed from the loci as reagents, immunoassays using antibodies to detect the levels of the encoded protein and a multiplicity of other methods dependent, for example, on other indicators of protein or mRNA levels, such as mass spectral patterns and chromatographic methods. The skilled practitioner will be aware of these and many other methods to detect expression levels.
  • immunoassays are a convenient embodiment.
  • antibodies specifically immunoreactive with the marker protein can be prepared by known procedures involving immunization of suitable subjects, such as mice, rabbits, or goats and preparation of polyclonal serum or, preferably, preparation of monoclonal forms of the antibodies using spleen or other sources of B-cells and standard fusions techniques.
  • genes encoding monoclonal antibodies may be isolated and used to produce recombinant forms, including modified forms such as Fv single-chain antibodies. Fragments of antibodies which are themselves immunoreactive with the target proteins can also be used in the assays.
  • the antibodies are used in in vitro diagnosis, there may be no reason to further modify, for example, murine antibodies to prepare human/murine chimeras or to humanize them for human diagnosis. However, use of such modified forms is not excluded.
  • antibodies includes the various forms of immunoglobulins and immunoreactive fragments of these proteins.
  • “antibodies” includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, fragments such as Fab, Fab', F(ab') 2 , single-chain antibodies (Fv), chimeric antibodies, antibodies recombinantly produced or antibodies produced from hybridomas, and the like. Humanized antibodies are also included, though their use in in vitro diagnosis is not required.
  • antibodies useful in detecting some of the marker proteins of the present invention. These antibodies recognize specific epitopes of the protein specifically bound by them.
  • the invention is also directed to these antibodies and to antibodies which bind the same epitopes as do these exemplified antibodies.
  • an antibody is 'immunoreactive' with a particular protein is meant that the antibody (or immunoreactive fragment) binds to that protein in a manner that permits its detection in a complex environment. It is understood that many antibodies are crossreactive with other proteins, but generally at levels much less than those with which they are specifically immunoreactive.
  • Immunoassays may he performed in many formats including direct and competitive sandwich assays, assays which utilize radioactive tracers as detection reagents, fluorescence detection, chemiluminescent detection, detection by complexation with enzymes (ELISA assays) or various forms of flow cytometry.
  • sandwich assays employs monoclonal antibodies in a sandwich assay and detection with labeled capping antibody.
  • the labeled capping antibody described is a purified rabbit polyclonal antibody but could be a mono or polyclonal antibody from a variety of sources.
  • a variety of other protocols could also be used, such as lateral flow assays, centrifugal flow assays, assay strips, homogeneous assays and the like.
  • kits useful for performing diagnosis If the assay is an immunoassay, the kit will include at least an antibody or fragment or modified form thereof which is immunoreactive with the designated marker. It may also include additional antibodies for detection of the resulting complex or other reagents for such detection. The assay kit may also include solid support for capture and labeling of the analyte.
  • Kits which depend on detection of mRNA will include specific primers designed to amplify the mRNA encoding the marker or an oligonucleotide probe which will hybridize thereto. Suitable detection reagents are also included.
  • any specific binding partner for the analyte expression product may be used - e.g ., a ligand for receptor protein and vice versa, or reagents specifically designed to bind target RNA or DNA generated therefrom may be employed.
  • RNA LabChip Agilent 2100 Bioanalyzer RNA 6000 LabChip kit.
  • Biotinylated cRNA probes were generated, fragmented and applied to Mouse MU74A (Version 1) GeneChip ® arrays (Affymetrix, Santa Clara, CA). The overall fluorescence intensity across each chip was scaled to 1500 with Affymetrix analysis software, Microarray Suite. The data were transferred to Microsoft Excel work sheet.
  • One selection criterion was gene expression with average difference values > 10,000 in the brain (i.e. , signal intensity of each mRNA computed by Affymetrix software that calculates the difference between the perfect match and mismatch oligonucleotides which are part of the gene chip array).
  • the average difference values as described above reflect an expression level above "background” and demonstrate sufficient abundance of the expression products to provide a suitable marker.
  • mRNA/protein products of the foregoing genes are thus predicted to be present in cerebrospinal fluid, blood or urine after brain injury or more generally as an indicator of brain damage.
  • FIG. 1A A graphical representation of the comparative expression levels of mRNA in fresh mouse brain as compared to other tissues is shown in Figures 1A and 1B for the final candidate genes shown in Table 1.
  • Nucleotide sequences encoding marker proteins were inserted into pGEX or pET Vectors for production of protein in E . coli.
  • Proteins from pET vectors were purified using Qiagen Ni-NTA following manufacturer's protocol (The Qiaexpressionist 06/2003; Qiaben, Valencia, CA).
  • Those from the pGEX vector were purified using immobilized glutathione from Pierce (Rockford, IL) following manufacturer's protocol. In some cases plasmids were sent to GenWay Biotech, Inc. (San Diego, CA) for large scale production of protein from E . coli.
  • pET 100 GAD 67 pET 102 GAD 67
  • pET 28 Zygin pET 28 Zygin I.
  • Quality control included analysis on SDS-PAGE, N-terminal Edman sequencing and Mass Spectrometry as appropriate ( Li, A., et al., (2003) Protein Expr Purif, 31(2):197-206 ). Protein concentration was estimated by Absorbance at 280 nm using calculated extinction coefficients from the protein sequence plus construct obtained at the Swiss-Prot web site and agreed with visual inspection of protein-stained bands on SDS-PAGE.
  • Anti-peptide immunogens were prepared as in Li, A., et al., 2003 ( supra ). Rabbits were immunized at Harland Bioproducts for Science, Inc. (Madison, WI). Rabbit serum was immunopurified over an affinity column containing the cognate peptide or protein antigen. Mice were immunized with 25 ug/mouse immunogen in MPL-TDM adjuvant (Sigma-Aldrich, St. Louis, MO) followed by at least 2 boosts in adjuvant and a 3 rd in PBS 3 days before fusion.
  • MPL-TDM adjuvant Sigma-Aldrich, St. Louis, MO
  • Subclass determination for monoclonal Ab's utilized the IsoStrip kit from Roche (Indianapolis, IN). Minimum epitope assignment was based on immunostaining of ABIMED spot peptide arrays prepared at the MIT Biopolymers Facility (Cambridge, MA). Each spot comprised a 10-mer contiguous peptide, and depending on the number of residues in the antigen of interest, either a one-, two-, or three-residue offset was used to cover the entire antigen sequence. For example, for a one-residue offset spot 1 contains sequence 1-10, spot 2 sequence 2-11, spot 3 sequence 3-12, etc.
  • Monoclonal antibodies were raised against VLP-1 using a combination of DNA and protein injections.
  • the vector VR1012 (Vical Inc.) and certain sequences from CTLA4Ig were used for the DNA injections.
  • VR1012 has been optimized for protein expression in mouse skeletal muscle, whereas, sequences contained in CTLA4Ig were previously shown to greatly increase the antibody response in mice ( Boyle, et al., Nature (1998) 392:408-411 ).
  • the presence of protein expressed by some of the candidate genes were assessed in various tissues.
  • a Western blot was performed on a human tissue array, (GenoTechnology, Inc. 50 ⁇ g per tissue).
  • the human tissue array included liver, brain, lung, kidney, spleen, testis, ovary, heart, pancreas, uterus, breast, cervix, rectum, prostate, thyroid, laryngopharynx, stomach, and skin.
  • human VLP-1 there is high protein expression in brain with far less in cervix and some in skin.
  • the lack of protein expression in the other tissues is consistent with the mRNA expression data.
  • neuroserpin some protein was found in prostate, and thyroid, but far less than brain, and a trace in kidney and pancreas.
  • the protein gene product encoded by SNAP-25 was found in abundance in brain with only trace amounts in a few other tissues. Likewise for the protein encoded by MOBP; only a very trace amount was found in pancreas. Zygin and GAD67 protein expression occurs only in brain tissue and not in any of the other tissues tested.
  • VLP-1 visininin like 1
  • MSD Meso Scale Discovery
  • Figures 4A and 4B show a typical standard curve generated via a 4-parameter curve-fitting software.
  • Figure 4A shows the range up to 12,500 ⁇ g/ml;
  • panel B shows an expanded depiction of the standard curve in the region from 0-1,250 ⁇ g/ml.
  • RLU refers to relative light units.
  • the sensitivity of the assay (lowest measurable amount) was estimated as 41.1 ⁇ g/ml based on the assay blank + 3 standard deviations.
  • a rat stroke model was employed to assess the time course of appearance of visinin-like 1 in the blood and cerebrospinal fluid, as a function of time.
  • pMCAO Permanent Middle Cerebral Artery Occlusion
  • the filament was advanced 17.5 mm to occlude the origin of the MCA.
  • the filament was secured in place in the right common carotid artery using a surgical nylon suture. After surgery, anesthesia (isoflurane) was discontinued and the animals were allowed to recover.
  • Blood was collected (approximately 0.25 ml) at 5 time-points (24 hours before surgery as well as 1, 4, 8, and 24 hours post-occlusion). The blood was allowed to clot at room temperature and subsequently centrifuged in order to collect serum samples. CSF was collected 24 hours post-occlusion. All samples were coded before assay, according to the following protocol.
  • Quantitative assays were also developed for neuroserpin, GAD67, and zygin.
  • the assay for neuroserpin utilized capture antibody 4221 5B5.1 (coated at 3 ⁇ g/ml) and 2 ⁇ g/ml biotinylated 4505 2F1.1. Detection was via streptavidin-alkaline phosphatase. (See Figure 7A .) Serpin was detected in 5 of 6 CSF samples from a rat model of ischemic stroke. The values ranged from 7 to 20 units of serpin.
  • a quantitative assay for GAD67 was developed utilizing electrochemiluminescence (MESO Scale Discovery).
  • the assay utilized immunopurified rabbit anti-GAD67 (R4043) as a capture antibody and Ruthenium labeled immunopurified rabbit anti-GAD67 (R4610) as a capping antibody.
  • the standard curve gives a signal value of about 1,000 units at a concentration of 1 ng/ml GAD67 and a non-linear progression to about 14,000 signal units at 100 ng/ml. (See Figure 7B .)
  • a quantitative assay for zygin was developed utilizing electrochemiluminescence (MESO Scale Discovery).
  • the assay uses monoclonal antibody 1G4.4 as a capture antibody (300 ng coated on to MSD plates) and Ruthenium labeled monoclonal antibody 4G3.1 as a detection antibody.
  • the standard curve ( Figure 7C ) provides a general linear relationship of between 1,000 MSD units at 1 ng/ml to about 3,500 MSD units at 3 ng/ml.
  • a human CSF postmortem sample (Analytical Biological Services, Wilmington, DE) was analyzed to determine if some of the brain markers would be found in CSF from brains deprived of oxygen for hours.
  • Table 3 shows a summary of the results set forth above as determined for certain of the marker proteins.
  • Table 3 Protein Expression of Some Brain-Specific Genes* Gene Western Blot of Human Brain Other Human Tissues Found in Human Blood After Ischemic Stroke Postmortem Human CSF Rat Stroke Model CSF VSNL1 + Cervix, skin Yes Yes Yes FEZ1 + None Yes SERPINI1 + Prostate, thyroid and trace in kidney and pancreas Yes GAD1 + None Yes INA + SNAP25 + Trace in a few other tissues MOBP + Trace in pancreas PLP1 Neg * Areas not filled in were not done.
  • the proteins expressed by the brain-specific genes are found in significant amounts in the brain and only slightly, if at all, in other tissues.
  • the proteins tested were also found in blood after stroke in humans or in blood and/or CSF in a rat model of stroke.
  • the protein products of the brain-specific genes give similar findings as the mRNA expression of the genes.

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Claims (10)

  1. Ein Verfahren zur Diagnose des Auftretens von Hirnschäden in einem Subjekt, das Verfahren umfassend das Analysieren einer Probe, die eine biologische Flüssigkeit umfasst, oder einer Fraktion davon, von dem Subjekt auf die Anwesenheit eines Markers, der die mRNA oder das Proteinprodukt des Gens ist, das auf Gen I.D. Nr. 7447 (VSNL1) lokalisiert ist und visinin-like 1 (VLP-1) kodiert, wobei die Anwesenheit des Markers Hirnschäden anzeigt.
  2. Das Verfahren nach Anspruch 1, wobei das Subjekt ein Mensch ist.
  3. Das Verfahren nach Anspruch 1 oder 2, wobei die Hirnschäden durch einen Schlaganfall oder die Alzheimer-Krankheit hervorgerufen werden.
  4. Das Verfahren nach einem der Ansprüche 1-3, wobei die biologische Flüssigkeit CSF, Blutserum, Urin oder Blutplasma ist.
  5. Das Verfahren nach Anspruch 4, wobei die biologische Flüssigkeit Blutserum oder Blutplasma ist.
  6. Das Verfahren nach einem der Ansprüche 1-5, wobei das Analysieren die Behandlung der Probenflüssigkeit mit mindestens einem Antikörper, der immunoreaktiv mit VLP-1 ist, umfasst.
  7. Das Verfahren nach Anspruch 6, wobei das monoklonale Protein ein Epitop, das aus Aminosäuren 3-11, 16-23 und 139-155 des humanen VLP-1 ausgewählt ist, erkennt.
  8. Das Verfahren nach Anspruch 6 oder 7, wobei der Antikörper monoklonal ist.
  9. Das Verfahren nach einem der Ansprüche 1-5, das ferner das Analysieren der Probe auf die Anwesenheit eines zusätzlichen Markers, der die mRNA oder das Proteinprodukt eines Gens ist, das aus der Gruppe bestehend aus solchen, die auf
    Gen I.D. Nr. 6616 (SNAP25), das das synaptosomal-assoziierte Protein, 25 kDa, kodiert,
    Gen I.D. Nr. 2571 (GAD1), das die Glutamat-decarboxylase 1, (Gehirn, 67 kDa) (GAD67) kodiert,
    Gen I.D. Nr. 4336 (MOBP), das das Myelin-assoziierte oligodendrozytische basische Protein kodiert,
    Gen I.D. Nr. 6857 (SYT1), das Synaptotagmin I kodiert,
    Gen I.D. Nr. 10382 (TUBB4), das Tubulin, beta 4 kodiert;
    Gen I.D. Nr. 9638 (FEZ1), das das Faszikulations- und Elongationsprotein, zeta 1 (zygin I), kodiert,
    Gen I.D. Nr. 2743 (GLRB), das den Glycin-Rezeptor, beta, kodiert,
    Gen I.D. Nr. 140767 (VMP), das das vesikulare Membranprotein p24 kodiert,
    Gen I.D. Nr. 10439 (OLFMI), das Olfactomedin 1 kodiert,
    Gen I.D. Nr. 7545 (ZIC1), das das Zic Familienmitglied 1 (odd-paired homolog, Drosophila) kodiert,
    Gen I.D. Nr. 29993 (PACSIN1), das das Protein-Kinase C und Casein-Kinase Substrat in Neuronen 1 kodiert,
    Gen I.D. Nr. 5354 (PLP1), das Proteolipidprotein 1 (Pelizaeus-Merzbacher Krankheit, spastische Paraplegie 2) kodiert,
    Gen I.D. Nr. 9118 (INA), das das internexin neuronale Intermediat-Filament Protein, alpha, kodiert,
    Gen I.D. Nr. 140679 (SLC32A1), das den gelösten Träger Familie 32 (GABA Vesikulartransporter) Mitglied 1 kodiert,
    Gen I.D. Nr. 5274 (SERPINI1), das den Serin (oder Cystein) Proteinaseinhibitor, Stamm 1 (neuroserpin), Mitglied 1, kodiert,
    Gen I.D. Nr. 4826 (NNAT), das Neuronatin kodiert,
    Gen I.D. Nr. 2566 (GABRG2), das den gamma-Aminobuttersäure (GABA) A Rezeptor, gamma 2, kodiert,
    Gen I.D. Nr. 6844 (VAMP2), das das Vesikel-assoziierte Membranprotein 2 (synaptobrevin T) kodiert oder
    Gen I.D. Nr. 4900 (NRGN), das Neurogranin (Protein-Kinase C Substrat, RC3) kodiert, lokalisiert sind, ausgewählt wird, beinhaltet.
  10. Das Verfahren nach Anspruch 9, wobei der zusätzliche Marker Neuroserpin ist und die Antikörper immunoreaktiv mit einem Epitop sind, das durch die Antikörper 4421 2G10.2; und/oder 4421 5B5.1; und/oder 4421 7D6.3; und/oder 4505 2F1.1 erkannt wird, oder wobei der zusätzliche Marker Zygin ist und die Antikörper immunoreaktiv mit einem Epitop sind, das durch die Antikörper 4554 IG4.4; und/oder 4563 4G3.1; und/oder R4726; und/oder R4727 erkannt wird, oder
    wobei der zusätzliche Marker GAD67 ist und die Antikörper immunoreaktiv mit einem Epitop sind, das durch die Antikörper R4610; und/oder R4609; und/oder R4043; und/oder R4404, und/oder Chemicon MAB5406 erkannt wird, oder
    wobei der zusätzliche Marker Intemexin ist und die Antikörper immunoreaktiv mit einem Epitop sind, das durch die Antikörper SC-7571 und/oder SC-7570 erkannt wird, oder
    wobei der zusätzliche Marker das synaptosomal-assoziierte Protein-25 kD ist und die Antikörper immunoreaktiv mit einem Epitop sind, das durch die Antikörper SC-7538;
    und/oder SC-7539 und/oder SC-20038 erkannt wird, oder
    wobei der zusätzliche Marker das Proteolipidprotein 1 ist und die Antikörper immunoreaktiv mit einem Epitop sind, das durch die Antikörper SC-23570; und/oder SC-18529 erkannt wird,
    oder
    wobei der zusätzliche Marker das Myelin-assoziierte oligodendrocytische basische Protein ist und die Antikörper immunoreaktiv mit einem Epitop sind, das durch die Antikörper SC-14250 und/oder SC-25666 erkannt wird.
EP05763857.9A 2004-06-25 2005-06-27 Marker für hirnschädigung Active EP1774023B1 (de)

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WO2009026432A1 (en) 2007-08-21 2009-02-26 Washington University Improved alzheimer's diagnosis
WO2012051519A2 (en) * 2010-10-14 2012-04-19 The Johns Hopkins University Biomarkers of brain injury
US20140141458A1 (en) 2011-05-12 2014-05-22 The Johns Hopkins University Assay reagents for a neurogranin diagnostic kit
WO2013138509A1 (en) 2012-03-13 2013-09-19 The Johns Hopkins University Citrullinated brain and neurological proteins as biomarkers of brain injury or neurodegeneration
WO2015009907A1 (en) 2013-07-17 2015-01-22 The Johns Hopkins University A multi-protein biomarker assay for brain injury detection and outcome
US9482675B1 (en) * 2013-07-31 2016-11-01 University Of Kentucky Research Foundation Methods and systems for prognosis and diagnosis of brain damage
CA2936056A1 (en) * 2014-01-06 2015-07-09 Children's Medical Center Corporation Biomarkers for dementia and dementia related neurological disorders
EP3242134A1 (de) 2016-05-04 2017-11-08 Euroimmun Medizinische Labordiagnostika AG Assay zur diagnose einer neurologischen krankheit
WO2018080514A1 (en) 2016-10-28 2018-05-03 University Of Kentucky Research Foundation Methods for detection and treatment of brain injury

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US20040018522A1 (en) * 2002-05-09 2004-01-29 Brigham And Women's Hospital, Inc. Identification of dysregulated genes in patients with multiple sclerosis
ATE500324T1 (de) * 2002-12-09 2011-03-15 Antonio Camargo Inhibitoren der enzymaktivität der endooligopeptidase a (eopa) und ihre verwendungen
ES2482142T3 (es) * 2004-04-15 2014-08-01 University Of Florida Research Foundation, Inc. Proteínas neurales como biomarcadores para lesiones del sistema nervioso y otros trastornos neurales

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US7985555B2 (en) 2011-07-26
US20080131881A1 (en) 2008-06-05
US20120021534A1 (en) 2012-01-26
CA2571445C (en) 2015-07-28
EP2514837A1 (de) 2012-10-24
EP1774023A4 (de) 2009-04-01
WO2006012351A2 (en) 2006-02-02
JP2008504032A (ja) 2008-02-14
EP1774023A2 (de) 2007-04-18
EP2514837B1 (de) 2016-02-10
US9982299B2 (en) 2018-05-29
ES2428795T3 (es) 2013-11-11
CA2571445A1 (en) 2006-02-02

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